This proposal is a competitive renewal. In the 3.5 years since we received funding, we have published 29 papers acknowledging support from this grant; another five papers are under review. These publications address issues in diagonal capillary electrophoresis, including reactor design and improved capillary zone electrophoresis separations. We and others have demonstrated that capillary zone electrophoresis consistently provides more protein and peptide identifications than high performance liquid chromatography for mass-limited samples. We will take advantage of our experience to develop and evaluate technology for the bottom-up proteomic analysis of single blastomeres. Our first aim will develop on-column sample preconcentration technology, coupled with capillary zone electrophoresis peptide separation and an electrokinetically-pumped sheath flow nanoelectrospray interface with tandem mass spectrometry detection. Our second aim will develop on-column cell lysis, reduction and alkylation, digestion, and preconcentration in an integrated device, which will be evaluated using blastomeres along the D1 lineage. Our third aim provides bioinformatic support and ensures wide distribution of our results. This project also builds off of a recent publication from this team, which reported the first quantitative proteomic analysis of a developing embryo (Sci Rep. 2014; 4: 4365. PMID: 24626130). We studied single Xenopus laevis embryos at six stages of development, including stage 1 embryos. These stage 1 embryos have not divided, and constitute a single cell; we quantified the expression of 4,000 proteins from these single cells. We will extend our Sci Rep publication by quantitatively monitoring protein expression in single blastomeres from stage 2 of development through stage 20. These cells contain from 50-g to 50-pg of protein, and form a natural progression of cells with progressively smaller protein content, ultimately ending at the size of a typical mammalian somatic cell. This progression of cell sizes is ideal for the development and evaluation of technology for single cell analysis. We will focus on the analysis of single cells isolated from the lineage of the blastomere D1, which ultimately form portions of the adult retina, spinal cord, and brain. Proteomic analysis of single blastomeres isolated from this lineage will provide insight into the assembly of the organism's nervous system.